Voltage-gated sodium channel scn8a is required for innervation and regeneration of amputated adult zebrafish fins.

Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab. Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.

Agrin/Lrp4 signal constrains MuSK-dependent neuromuscular synapse development in appendicular muscle.

The receptor tyrosine kinase MuSK, its co-receptor Lrp4 and the Agrin ligand constitute a signaling pathway that is crucial in axial muscle for neuromuscular synapse development, yet whether this pathway functions similarly in appendicular muscle is unclear. Here, using the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, we show that, similar to axial muscle, developing appendicular muscles form aneural acetylcholine receptor (AChR) clusters prior to innervation. As motor axons arrive, neural AChR clusters form, eventually leading to functional synapses in a MuSK-dependent manner. We find that loss of Agrin or Lrp4 function, which abolishes synaptic AChR clusters in axial muscle, results in enlarged presynaptic nerve regions and progressively expanding appendicular AChR clusters, mimicking the consequences of motoneuron ablation. Moreover, musk depletion in lrp4 mutants partially restores synaptic AChR patterning. Combined, our results provide compelling evidence that, in addition to the canonical pathway in which Agrin/Lrp4 stimulates MuSK activity, Agrin/Lrp4 signaling in appendicular muscle constrains MuSK-dependent neuromuscular synapse organization. Thus, we reveal a previously unappreciated role for Agrin/Lrp4 signaling, thereby highlighting distinct differences between axial and appendicular synapse development.

Neuronal Circuits That Control Rhythmic Pectoral Fin Movements in Zebrafish.

The most basic form of locomotion in limbed vertebrates consists of alternating activities of the flexor and extensor muscles within each limb coupled with left/right limb alternation. Although larval zebrafish are not limbed, their pectoral fin movements exhibit the following fundamental aspects of this basic movement: abductor/adductor alternation (corresponding to flexor/extensor alternation) and left/right fin alternation. Because of the simplicity of their movements and the compact neural organization of their spinal cords, zebrafish can serve as a good model to identify the neuronal networks of the central pattern generator (CPG) that controls rhythmic appendage movements. Here, we set out to investigate neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish, using transgenic fish that specifically express GFP in abductor or adductor motor neurons (MNs) and candidate CPG neurons. First, we showed that spiking activities of abductor and adductor MNs were essentially alternating. Second, both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively, indicating that the MN spiking activities are controlled in a push-pull manner. Further, we obtained the following evidence that dmrt3a-expressing commissural inhibitory neurons are involved in regulating the activities of abductor MNs: (1) strong inhibitory synaptic connections were found from dmrt3a neurons to abductor MNs; and (2) ablation of dmrt3a neurons shifted the spike timing of abductor MNs. Thus, in this simple system of abductor/adductor alternation, the last-order inhibitory inputs originating from the contralaterally located neurons play an important role in controlling the firing timings of MNs.SIGNIFICANCE STATEMENT Pectoral fin movements in larval zebrafish exhibit fundamental aspects of basic rhythmic appendage movement: alternation of the abductor and adductor (corresponding to flexor-extensor alternation) coupled with left-right alternation. We set out to investigate the neuronal circuits underlying rhythmic pectoral fin movements in larval zebrafish. We showed that both abductor and adductor MNs received rhythmic excitatory and inhibitory synaptic inputs in their active and inactive phases, respectively. This indicates that MN activities are controlled in a push-pull manner. We further obtained evidence that dmrt3a-expressing commissural inhibitory neurons exert an inhibitory effect on abductor MNs. The current study marks the first step toward the identification of central pattern generator organization for rhythmic fin movements.

Indirect pathway to pectoral fin motor neurons from nucleus ruber in the Nile tilapia Oreochromis niloticus.

Supraspinal motor control systems of pectoral fins remain unclear in teleosts. Nucleus ruber of Goldstein (1905; NRg), which has been identified as the probable homologue of nucleus ruber of tetrapods, is a candidate structure serving for such functions. In the present study, we investigated possible involvement of the NRg in the control of pectoral fin movement by tract-tracing experiments in the Nile tilapia Oreochromis niloticus. Tracer injections into the NRg revealed the fiber course of rubrospinal tract. Rubrospinal fibers crossed the midline at the level of midbrain, descended through the tegmentum, and terminated in a region ventrally adjacent to the dorsal horn at the spinomedullary junction, without reaching the ventral horn where pectoral fin motor neurons are present. Tracer injection experiments into the dorsal horn region resulted in labeled terminals in proximities of presumed pectoral fin motor neurons in the ventral horn. Tracer injection experiments into the ventral horn resulted in retrogradely labeled neurons ventrally adjacent to the dorsal horn, where labeled terminals were detected following rubral injections. These anatomical analyses suggest that the NRg of actinopterygians is involved in the control of pectoral fin motor neurons through an indirect pathway via interneurons in the dorsal horn.

New details of the neural architecture of the salmonid adipose fin.

The adipose fin of salmonids, once widely regarded as vestigial and lacking in function, was shown to be important to swimming efficiency in juvenile brown trout Salmo trutta. Examination with confocal microscopy of adipose fins of S. trutta stained with various antibodies targeting the nervous system revealed several large nerves entering the fin and anastomosing throughout its length. The branching nerves form a plexus with specific patterns of fine terminal branches in the leading and trailing edges. A network of astrocyte-like cells (ALCs) that is linked through cell processes to nerves and structural collagen reacted positively with antibodies to glial cells. No other fish fins, including other adipose fins, have been shown to exhibit this type of neural architecture. Many vertebrate mechanoreceptors rely on collagen deformation to stimulate responses in afferent nerves; similarly, the adipose fin also may function as a mechanosensor, where passive mechanical deflection by water currents stimulates afferent nerves.

Paclitaxel-induced epithelial damage and ectopic MMP-13 expression promotes neurotoxicity in zebrafish.

Paclitaxel is a microtubule-stabilizing chemotherapeutic agent that is widely used in cancer treatment and in a number of curative and palliative regimens. Despite its beneficial effects on cancer, paclitaxel also damages healthy tissues, most prominently the peripheral sensory nervous system. The mechanisms leading to paclitaxel-induced peripheral neuropathy remain elusive, and therapies that prevent or alleviate this condition are not available. We established a zebrafish in vivo model to study the underlying mechanisms and to identify pharmacological agents that may be developed into therapeutics. Both adult and larval zebrafish displayed signs of paclitaxel neurotoxicity, including sensory axon degeneration and the loss of touch response in the distal caudal fin. Intriguingly, studies in zebrafish larvae showed that paclitaxel rapidly promotes epithelial damage and decreased mechanical stress resistance of the skin before induction of axon degeneration. Moreover, injured paclitaxel-treated zebrafish skin and scratch-wounded human keratinocytes (HEK001) display reduced healing capacity. Epithelial damage correlated with rapid accumulation of fluorescein-conjugated paclitaxel in epidermal basal keratinocytes, but not axons, and up-regulation of matrix-metalloproteinase 13 (MMP-13, collagenase 3) in the skin. Pharmacological inhibition of MMP-13, in contrast, largely rescued paclitaxel-induced epithelial damage and neurotoxicity, whereas MMP-13 overexpression in zebrafish embryos rendered the skin vulnerable to injury under mechanical stress conditions. Thus, our studies provide evidence that the epidermis plays a critical role in this condition, and we provide a previously unidentified candidate for therapeutic interventions.

Fin ray sensation participates in the generation of normal fin movement in the hovering behavior of the bluegill sunfish (Lepomis macrochirus).

For many fish species, the pectoral fins serve as important propulsors and stabilizers and are precisely controlled. Although it has been shown that mechanosensory feedback from the fin ray afferent nerves provides information on ray bending and position, the effects of this feedback on fin movement are not known. In other taxa, including insects and mammals, sensory feedback from the limbs has been shown to be important for control of limb-based behaviors and we hypothesized that this is also the case for the fishes. In this study, we examined the impact of the loss of sensory feedback from the pectoral fins on movement kinematics during hover behavior. Research was performed with bluegill sunfish (Lepomis macrochirus), a model for understanding the biomechanics of swimming and for bio-inspired design of engineered fins. The bluegill beats its pectoral fins rhythmically, and in coordination with pelvic and median fin movement, to maintain a stationary position while hovering. Bilateral deafferentation of the fin rays results in a splay-finned posture where fins beat regularly but at a higher frequency and without adducting fully against the side of the body. For unilateral transections, more irregular changes in fin movements were recorded. These data indicate that sensory feedback from the fin rays and membrane is important for generating normal hover movements but is not necessary for generating rhythmic fin movement.

V-ATPase proton pumping activity is required for adult zebrafish appendage regeneration.

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration.

Denervation impairs regeneration of amputated zebrafish fins.

BACKGROUND: Zebrafish are able to regenerate many of its tissues and organs after damage. In amphibians this process is regulated by nerve fibres present at the site of injury, which have been proposed to release factors into the amputated limbs/fins, promoting and sustaining the proliferation of blastemal cells. Although some candidate factors have been proposed to mediate the nerve dependency of regeneration, the molecular mechanisms involved in this process remain unclear. RESULTS: We have used zebrafish as a model system to address the role of nerve fibres in fin regeneration. We have developed a protocol for pectoral fin denervation followed by amputation and analysed the regenerative process under this experimental conditions. Upon denervation fins were able to close the wound and form a wound epidermis, but could not establish a functional apical epithelial cap, with a posterior failure of blastema formation and outgrowth, and the accumulation of several defects. The expression patterns of genes known to be key players during fin regeneration were altered upon denervation, suggesting that nerves can contribute to the regulation of the Fgf, Wnt and Shh pathways during zebrafish fin regeneration. CONCLUSIONS: Our results demonstrate that proper innervation of the zebrafish pectoral fin is essential for a successful regenerative process, and establish this organism as a useful model to understand the molecular and cellular mechanisms of nerve dependence, during vertebrate regeneration.

Neural network detected in a presumed vestigial trait: ultrastructure of the salmonid adipose fin.

A wide variety of rudimentary and apparently non-functional traits have persisted over extended evolutionary time. Recent evidence has shown that some of these traits may be maintained as a result of developmental constraints or neutral energetic cost, but for others their true function was not recognized. The adipose fin is small, fleshy, non-rayed and located between the dorsal and caudal fins on eight orders of basal teleosts and has traditionally been regarded as vestigial without clear function. We describe here the ultrastructure of the adipose fin and for the first time, to our knowledge, present evidence of extensive nervous tissue, as well as an unusual subdermal complex of interconnected astrocyte-like cells equipped with primary cilia. The fin contains neither adipose tissue nor fin rays. Many fusiform actinotrichia, comprising dense striated macrofibrils, support the free edge and connect with collagen cables that link the two sides. These results are consistent with a recent hypothesis that the adipose fin may act as a precaudal flow sensor, where its removal can be detrimental to swimming efficiency in turbulent water. Our findings provide insight to the broader themes of function versus constraints in evolutionary biology and may have significance for fisheries science, as the adipose fin is routinely removed from millions of salmonids each year.

Hydrogen peroxide promotes injury-induced peripheral sensory axon regeneration in the zebrafish skin.

Functional recovery from cutaneous injury requires not only the healing and regeneration of skin cells but also reinnervation of the skin by somatosensory peripheral axon endings. To investigate how sensory axon regeneration and wound healing are coordinated, we amputated the caudal fins of zebrafish larvae and imaged somatosensory axon behavior. Fin amputation strongly promoted the regeneration of nearby sensory axons, an effect that could be mimicked by ablating a few keratinocytes anywhere in the body. Since injury produces the reactive oxygen species hydrogen peroxide (H(2)O(2)) near wounds, we tested whether H(2)O(2) influences cutaneous axon regeneration. Exposure of zebrafish larvae to sublethal levels of exogenous H(2)O(2) promoted growth of severed axons in the absence of keratinocyte injury, and inhibiting H(2)O(2) production blocked the axon growth-promoting effects of fin amputation and keratinocyte ablation. Thus, H(2)O(2) signaling helps coordinate wound healing with peripheral sensory axon reinnervation of the skin.

Evolution of motor innervation to vertebrate fins and limbs.

The evolution and diversification of vertebrate behaviors associated with locomotion depend highly on the functional transformation of paired appendages. Although the evolution of fins into limbs has long been a focus of interest to scientists, the evolution of neural control during this transition has not received much attention. Recent studies have provided significant progress in the understanding of the genetic and developmental bases of the evolution of fin/limb motor circuitry in vertebrates. Here we compare the organization of the motor neurons in the spinal cord of various vertebrates. We also discuss recent advances in our understanding of these events and how they can provide a mechanistic explanation for the evolution of fin/limb motor circuitry in vertebrates.

Electron tomographic analysis of gap junctions in lateral giant fibers of crayfish.

Innexin-gap junctions in crayfish lateral giant fibers (LGFs) have an important role in escape behavior as a key component of rapid signal transduction. Knowledge of the structure and function of characteristic vesicles on the both sides of the gap junction, however, is limited. We used electron tomography to analyze the three-dimensional structure of crayfish gap junctions and gap junctional vesicles (GJVs). Tomographic analyses showed that some vesicles were anchored to innexons and almost all vesicles were connected by thin filaments. High densities inside the GJVs and projecting densities on the GJV membranes were observed in fixed and stained samples. Because the densities inside synaptic vesicles were dependent on the fixative conditions, different fixative conditions were used to elucidate the molecules included in the GJVs. The projecting densities on the GJVs were studied by immunoelectron microscopy with anti-vesicular monoamine transporter (anti-VMAT) and anti-vesicular nucleotide transporter (anti-VNUT) antibodies. Some of the projecting densities were labeled by anti-VNUT, but not anti-VMAT. Three-dimensional analyses of GJVs and excitatory chemical synaptic vesicles (CSVs) revealed clear differences in their sizes and central densities. Furthermore, the imaging data obtained under different fixative conditions and the immunolabeling results, in which GJVs were positively labeled for anti-VNUT but excitatory CSVs were not, support our model that GJVs contain nucleotides and excitatory CSVs do not. We propose a model in which characteristic GJVs containing nucleotides play an important role in the signal processing in gap junctions of crayfish LGFs.

No role for direct touch using the pectoral fins, as an information gathering strategy in a blind fish.

Blind Mexican cave fish (Astyanax fasciatus) lack a functional visual system and have been shown to sense their environment using a technique called hydrodynamic imaging, whereby nearby objects are detected by sensing distortions in the flow field of water around the body using the mechanosensory lateral line. This species has also been noted to touch obstacles, mainly with the pectoral fins, apparently using this tactile information alongside hydrodynamic imaging to sense their surroundings. This study aimed to determine the relative contributions of hydrodynamic and tactile information during wall following behaviour in blind Mexican cave fish. A wall was custom built with a 'netted' region in its centre, which provided very similar tactile information to a solid tank wall, but was undetectable using hydrodynamic imaging. The fish swam significantly closer to and collided more frequently with the netted region of this wall than the solid regions, indicating that the fish did not perceive the netted region as a solid obstacle despite being able to feel it as such with their pectoral fins. We conclude that the touching of objects with the pectoral fins may be an artefact of the intrinsic link between pectoral fin extensions and tail beating whilst swimming, and does not function to gather information. During wall following, hydrodynamic information appears to be used strongly in preference to tactile information in this non-visual system.

Ancestry of motor innervation to pectoral fin and forelimb.

Motor innervation to the tetrapod forelimb and fish pectoral fin is assumed to share a conserved spinal cord origin, despite major structural and functional innovations of the appendage during the vertebrate water-to-land transition. In this paper, we present anatomical and embryological evidence showing that pectoral motoneurons also originate in the hindbrain among ray-finned fish. New and previous data for lobe-finned fish, a group that includes tetrapods, and more basal cartilaginous fish showed pectoral innervation that was consistent with a hindbrain-spinal origin of motoneurons. Together, these findings support a hindbrain-spinal phenotype as the ancestral vertebrate condition that originated as a postural adaptation for pectoral control of head orientation. A phylogenetic analysis indicated that Hox gene modules were shared in fish and tetrapod pectoral systems. We propose that evolutionary shifts in Hox gene expression along the body axis provided a transcriptional mechanism allowing eventual decoupling of pectoral motoneurons from the hindbrain much like their target appendage gained independence from the head.